Use of L-α-lysophosphatidylcholine to obtain the differentiation of monocytes into mature dendritic cells in vitro

ABSTRACT

The present invention relates to the use of L-α-lysophosphatidylcholine and/or an equivalent compound for the differentiation of monocytes into mature denditric cell. 
     The present invention also relates to a method for differentiation monocytes into mature denditric cells according to which monocytes are provided in a medium suitable for their differentiation and L-α-lysophosphatidylcholine and/or an equivalent compound is added to said medium.

The present invention relates to a method for differentiating monocytesinto mature dendritic cells, according to which monocytes are providedin a suitable medium for their differentiation, andL-α-lysophosphatidylcholine is added to said medium. The presentinvention also relates to the use of at least one inhibitor ofL-α-lysophosphatidylcholine, for producing a medicinal product forpreventing an inflammation and/or for combating an inflammatory diseaseand/or an autoimmune disease.

Dendritic cells are involved in the development of an immune responseand in the initiation of a specific T-lymphocyte response byrecognition, uptake and presentation of antigens, in particular ofinfectious agents (Steinman et al. 1997, Immuno. Rev., 156: 25-37; Cellaet al. 1997, Curr. Opin. Immunol., 9: 10-16). While immature dendriticcells take up and digest the antigens of infectious agents veryeffectively, certain signals, such as bacterial agents or inflammatorycytokines, can activate them by inducing a process of maturation, whichis the initial step in triggering the adaptive immune response. In fact,during this activation, dendritic cells acquire the ability to migrateto lymphoid organs where T lymphocytes are found and the ability totransmit costimulation signals that are essential to the activation ofnaïve T lymphocytes. During this maturation, dendritic cells undergofunctional and phenotypic modifications such as:

-   -   an increase in surface molecules involved in the activation of T        lymphocytes (such as CD40, CD80, CD83, CD86 and the molecules of        the major histocompatibility complex (MHC) class I and class        II),    -   the production of proinflammatory cytokines (such as the        interleukins IL-12, IL-1β, TNFα and IL-6),    -   a decrease in their ability to take up and process the antigen.

By virtue of their abilities to develop an immune response and toinitiate a specific T lymphocyte response, mature dendritic cells are ofparticular therapeutic interest, in particular in the fields ofanti-infectious and antitumor immunization and of immunotherapy (Austin.1998, Curr. Opin. Hematol. 5: 3-15; Reise Sousa et al. 1999, Curr. Opin.Immunol. 11: 392-399). These dendritic cells, by virtue of their abilityto induce immuniotolerance, are also an advantageous target when it isdesired to inhibit the immune response of a patient, in particular inorder to combat autoimmune or inflammatory diseases.

In vitro, mature dendritic cells can be obtained from monocytes inculture. These monocytes are, in vivo, circulating cells which, whencrossing in particular the vascular endothelial wall, come into contactwith surrounding factors which influence their outcome in a manner whichis still poorly understood. Schematically, three possibilities are thenenvisioned for these monocytes:

-   -   exiting the tissues and returning to the lymph nodes,    -   differentiating into macrophages    -   differentiating into immature dendritic cells.

The first step for obtaining mature dendritic cells from monocytes inculture then consists in inducing the differentiation of the monocytesinto immature dendritic cells with, in particular, the interleukin IL-4and the factor GM-CSF (granulocyte macrophage-stimulating factor). After6 days, 95% of the cells in culture are immature dendritic cells.

The second step then consists of induction of the maturation of theimmature dendritic cells into mature dendritic cells using exogenousagents such as bacterial or viral agents. Thus, the KpOmpA protein fromKlebsiella pneumoniae is capable of inducing the maturation of theseimmature dendritic cells into mature dendritic cells (P. Jeannin et al.,Nature Immunology, 2000, 1: 502-509). Mention may also be made of thematuration of immature dendritic cells into mature dendritic cells bymeans of other exogenous molecules, such as bacterial membranelipopolysaccharides (Dichman et al, Journal of Cellular Physiology,2000, 185: 394-400). However, the use of exogenous molecules derivedfrom infectious agents induces problems of safety and of cost (direct orindirect side effects in vivo; very important need for purificationaccording to very strict legal or regulatory requirements, etc.), makingthe use of this type of molecule difficult in the context of vaccinologyand of immunotherapy.

More recently, it has been described, by Perrin-Cocon et al., in 2001(The Journal of Immunology, 167: 3785-3791), that the production ofmature dendritic cells from monocytes undergoing differentiation can beinduced with endogenous molecules, such as certain oxidized plasmalipoproteins, and more particularly oxidized low density lipoproteins(LDLs). However, oxidized LDLs are complex particles made up ofproteins, triacyl glycerols, phospholipids, and free and esterifiedcholesterol, and are as a result difficult to synthesize artificially.

It is also important to note that, while stimulation of thedifferentiation of monocytes into dendritic cells is essential incertain therapeutic applications (during immunization, duringstimulation of the immune response), it may also be essential to inhibitsuch a differentiation of monocytes into dendritic cells, or moregenerally to inhibit the maturation of immature dendritic cells, inother therapeutic applications, such as in an autoimmune or inflammatorydisease. Treatments for combating inflammation, such as the taking ofcorticoids or the taking of aspirin, currently exist. The problem isthat, when the inflammation is chronic, such treatments have sideeffects that are very harmful for the patient. The prolonged taking ofcorticoids can in particular engender a Cushing's syndrome during whichdemineralization, spontaneous fractures and diabetes are observed. Theprolonged taking of aspirin can, for its part, engender stomach ulcers.The present invention proposes to solve the disadvantages of the stateof the art by also proposing an anti-inflammatory molecule which is easyto synthesize and relatively inexpensive, and which inhibits thedifferentiation of monocytes into mature dendritic cells.

Thus, the present invention proposes to solve the disadvantages of thestate of the art by providing a proinflammatory molecule, an endogenousmolecule, which is easy to synthesize and relatively inexpensive andwhich stimulates the differentiation of monocytes into mature dendriticcells. The present invention also relates to the use of a lipid emulsioncomprising triglycerides, phospholipids and glycerol, such as inparticular INTRALIPID®, for inhibiting the differentiation of monocytesto mature dendritic cells, by virtue of a direct action, or an indirectaction via inhibition of the action of L-α-lysophosphatidylcholine.

Surprisingly, the present invention relates to the use ofL-α-lysophosphatidylcholine for differentiating monocytes into maturedendritic cells in vitro.

The term “L-α-lysophosphatidylcholine” is intended to mean a moleculefor which the formula is as follows:

in which

represents a long chain of saturated fatty acids containing from 12 to20 carbon atoms, preferably from 16 to 18.

In fact, one of the active molecules generated during LDL oxidation isL-α-lysophosphatidylcholine, hereinafter referred to as LPC. However, ithas been shown that the binding of LPC to the G2A receptor, which is thehigh affinity receptor for LPC, inhibits in particular the proliferationand the activation of T lymphocytes, rather suggesting a role for LPC ininhibiting the triggering of an immune reaction (Carson & Lo, 2001,Science, 293: 618-619). In addition, it has also been shown that a highconcentration of LPC (50 μM) inhibits the activity of the transcriptionfactor NF-κB (nuclear factor κB), which is however known to be activatedduring the maturation of dendritic cells. In addition, LPC is present athigh concentration in the plasma, suggesting that it is not active inthe plasma, which would not predispose it to be chosen by those skilledin the art for therapeutic use.

The invention relates to a method for the differentiation in vitro ofmonocytes into mature dendritic cells, according to which:

-   A. monocytes are provided in a suitable culture medium,-   B. the differentiation of the monocytes into dendritic cells is    induced in the presence of a differentiation factor,-   C. L-α-lysophosphatidylcholine is added to said medium and mature    dendritic cells are obtained.

The expression “suitable culture medium” is intended to mean a mediumcomprising all the elements required for cell viability. By way ofexample, mention may be made of RPMI 1640 medium and its derivatives,and any culture medium well known to those skilled in the art. Thismedium comprises in particular at least one factor for differentiatingmonocytes into dendritic cells. The factors for differentiatingmonocytes into dendritic cells are well known to those skilled in theart, and mention may in particular be made of cytokines such as, withoutany implied limitation, the interleukin IL-4, the factor GM-CFS(granulocyte macrophage-stimulating factor), IL-13 or TNF (tumornecrosis factor).

In step C), the L-α-lysophosphatidylcholine is added in particular tosaid medium at a final concentration in the medium of between 10 and 80μM, preferably between approximately 20 and 60 μM, and advantageouslybetween 30 and 50 μM. Still in step C), the L-α-lysophosphatidylcholineis added to said medium between the 3rd and 6th day of monocytedifferentiation, preferably between the 4th and 5th day of monocytedifferentiation.

According to a particular embodiment of the invention, at least onebiological agent is also added to the culture medium, in step C). Theterm “biological agent” is intended to mean a molecule (or a set ofmolecules) which is the target of an immune response or which allows thesynthesis of this target. This biological agent can thus be chosen frombacterial, viral, yeast, parasite or fungal antigens, tumor antigens,and lysates of autologous and/or heterologous tumor cells. The term“autologous tumor cells” is intended to mean tumor cells belonging tothe individual who receives a given medicinal product. The tumor cellscan be obtained by taking a sample of cancerous tissue, in particular abiopsy or a surgical resection. The term “heterologous tumor cells ” isintended to mean cells derived from tumors originating from anindividual who is different from the one receiving a given medicinalproduct. The use of heterologous cells makes it possible in particularto obtain a medicinal product for treating patients suffering fromcancer from whom it is not possible to obtain a tumor cell sample. Thisalso makes it possible to use a standard source of tumor antigens. Theterm “cell lysate” is intended to mean a mixture of intracellular and/ormembrane antigens, obtained by lysis of cells according to a protocolknown to those skilled in the art, such as mechanical, chemical orenzymatic lysis. This biological agent may also be a nucleic acid whichencodes at least one antigen chosen from bacterial, viral, yeast,parasite or fungal antigens, and tumor antigens. The term “tumorantigen” is intended to mean an antigen derived from tumor cells, suchas a tumor-related peptide, in particular a peptide which interacts withclass I molecules and which is presented to CD8 T lymphocytes. Mentionmay be made, in a known nonlimiting manner, of the following tumorantigens: MAGE-2, MAGE-3, MART, MUC-1, MUC-2, HER-2, GD2,carcinoembryonic antigen (CEA), TAG-72, ovarian-associated antigensOV-TL3 and MOV18, TUAN, alpha-fetoprotein (AEP), OFP, CA-125, CA-50,CA-19-9, renal tumor-associated antigen G250, EGP-40 (or EpCAM), S 100(malignant melanoma-associated antigen), p53, prostate tumor-associatedantigens (e.g. PSA and PSMA) and p21ras.

Thus, the addition of this biological agent to the culture medium makesit possible to obtain mature dendritic cells which then allow theactivation of T lymphocytes which are directed against a given antigen.These mature dendritic cells, obtained in vitro, can then be reinjectedin vivo.

In another embodiment of the invention, it is also possible to use, instep C, a compound equivalent to L-α-lysophosphatidylcholine, which is amolecule that acts according to the same cellular mechanisms of actionas L-α-lysophosphatidylcholine, i.e. by the same membrane receptors, inparticular G protein-coupled membrane receptors, such as the receptorsG2A, GPR4 or the PAF (platelet activating factor) receptor, and/or viathe same nuclear receptors, such as the PPAR receptors (peroxisomeproliferator-activated receptor). This equivalent compound can thus inparticular be an agonist for the abovementioned receptors (such as inparticular O-methyl-PAF, carbamyl-PAF or 2-O-methyl-PAF), but also thePAF itself. This compound may also be a PPARδ agonist, such as inparticular L165041 (Merck Research), GW-5015616 (Glaxo) orcarboprostacylin (Cayman), or a PPARγ antagonist, such as in particularbisphenol A diglycidyl ethyl (Sigma) or DICLOFENAC®(2-[(2,6-dichlorophenyl)amino ]benzenacetic acid). This compoundequivalent to L-α-lysophosphatidylcholine can be used alone or insynergy with L-α-lysophosphatidylcholine. According to a preferredembodiment of the invention, the L-α-lysophosphatidylcholine is used insynergy with platelet activating factor (PAF).

The invention also relates to a method for activating T lymphocytes invitro, according to which:

-   A. monocytes are provided in a suitable culture medium,-   B. the differentiation of the monocytes into dendritic cells is    induced in the presence of a differentiation factor,-   C. L-α-lysophosphatidylcholine is added to said medium and mature    dendritic cells are obtained,-   D. a biological agent, as defined above, is added to said medium and    the mature dendritic cells obtained in step B are directed against    said biological agent,-   E. the mature dendritic cells directed against the biological agent    according to step C are brought into contact with T lymphocytes, and    T lymphocytes directed against the biological agent are obtained.

Step C) can be carried out as described above. Such a method thus makesit possible to obtain, in vitro, T lymphocytes directed against a givenbiological agent, which can then be reinjected in vivo into a patient,in particular an immunodepressed patient.

The invention also relates to a method for maturing dendritic cells invitro, according to which:

-   A. immature dendritic cells are provided in a suitable culture    medium,-   B. L-α-lysophosphatidylcholine is added to said medium and mature    dendritic cells are obtained.

Those skilled in the art are well aware of dendritic cells and theirvarious stages of maturation.

In step B), the L-α-lysophosphatidylcholine is added in particular tosaid medium at a final concentration in the medium of betweenapproximately 10 and 80 μM, preferably between approximately 20 and 60μmM, and advantageously between 30 and 50 μM. According to a particularembodiment of the invention, at least one biological agent as definedabove is also added to the culture medium, in step B).

The invention also relates to a culture medium, characterized in that itcomprises L-α-lysophosphatidylcholine and at least one differentiationfactor as defined above.

In a preferred embodiment of the invention, L-α-lysophosphatidylcholineis at a final concentration in the medium of between approximately 10and 80 μM, preferably between approximately 20 and 60 μM, andadvantageously between 30 and 50 μM. According to a particularembodiment of the invention, the culture medium also comprises abiological agent as defined above, which is in particular a bacterial,viral, yeast, parasite or fungal antigen, an autologous and/orheterologous tumor cell lysate antigen, a tumor antigen, a nucleic acidwhich encodes a bacterial, viral, yeast, parasite or fungal antigen, ora nucleic acid which encodes a tumor antigen.

The invention also relates to the use of L-α-lysophosphatidylcholine asan agent for activating the immune system. The term “agent foractivating” is intended to mean a molecule which, in a pharmaceuticalcomposition, induces the effects of a medication or reinforces orcompletes the effects of the main medication. In the case of a vaccinecomposition, the L-α-lysophosphatidylcholine then plays the role ofadjuvant which stimulates the host organism's immune response against agiven antigen. Thus, the invention relates to a vaccine composition,characterized in that it comprises L-α-lysophosphatidylcholine as anagent for activating the immune system, which then plays the role ofadjuvant, and a biological agent as defined above, against which it isdesired to stimulate the patient's immune response.

According to a preferred embodiment of the invention,L-α-lysophosphatidylcholine is used as an agent for activating theimmune system, for producing a medicinal product for the treatmentand/or the prevention of an infection of bacterial, viral, fungal orparasitic origin or an infection caused by a yeast, and/or for theproduction of a medicinal product for the treatment and/or theprevention of cancers.

As bacterial infection, mention may in particular be made of infectionsinduced by staphylococci, mycobacteria, bacteria of the Nisseria genus,legionellae, salmonellae, etc.

As viral infection, mention may in particular be made of infectionsinduced by HIV (human immunodeficiency virus), hepatitis viruses, themeasles virus, the rubella virus, polio viruses, flavin viruses, etc.

As fungal infection, mention may in particular be made of aspergillosis,candidosis, etc.

As parasitic infection, mention may in particular be made of malaria,leishmaniasis, etc.

The term “cancer” is intended to mean all diseases due to an abnormalmultiplication of cells, and in particular, in a nonlimiting manner,melanomas, lymphomas, leukemias, kidney, brain, colon, prostate, rectal,pancreatic, ovarian, lung, liver and breast carcinomas, skin cancerschosen from keratinomas and carcinomas, and melanomas.

The medicinal product according to the invention may be provided in theform of a pharmaceutical composition in combination with at least onepharmaceutically acceptable excipient well known to those skilled in theart. In the pharmaceutical compositions according to the invention, fororal, sublingual, subcutaneous, intramuscular, intravenous, topical,intratracheal, rectal or transdermal administration, theL-α-lysophosphatidylcholine can be administered in unit administrationform or as a mixture with conventional pharmaceutical supports, and whenintended for oral administration, for example in the form of a tablet, agel capsule, an oral solution, etc., or for rectal administration, inthe form of a suppository, for parenteral administration, in particularin the form of an injectable solution, especially by intravenous,intradermal or subcutaneous injection, etc., according to conventionalprotocols well known to those skilled in the art. For topicalapplication, the L-α-lysophosphatidylcholine can be used in creams,ointments, lotions or eye lotions.

When a solid composition is prepared in the form of tablets, theL-α-lysophosphatidylcholine is mixed with a pharmaceutically acceptableexcipient, also called pharmaceutical vehicle, such as gelatin, starch,lactose, magnesium stearate, talc, gum arabic or the like. The tabletsmay be coated with sucrose, with a cellulose derivative or with othersuitable materials. They can also be treated such that they haveprolonged or delayed activity and such that they continuously release apredetermined amount of L-α-lysophosphatidylcholine. It is also possibleto obtain a preparation of gel capsules by mixing theL-α-lysophosphatidylcholine with a diluent and pouring the mixture intosoft or hard gel capsules. It is also possible to obtain a preparationin the form of syrup or for administration in the form of drops, inwhich the L-α-lysophosphatidylcholine is present together with asweetener, an antiseptic, such as in particular methylparaben andpropylparaben, and also a suitable flavor enhancer or dye.Water-dispersible powders or granules can contain theL-α-lysophosphatidylcholine as a mixture with dispersing agents orwetting agents, or suspending agents, well known to those skilled in theart. For parenteral administration, use is made of aqueous suspensions,isotonic saline solutions or sterile and injectable solutions whichcontain dispersing agents or wetting agents which are pharmacologicallycompatible, such as in particular propylene glycol or butylene glycol.

The invention also relates to the use of at least one inhibitor ofL-α-lysophosphatidylcholine, for producing a medicinal product forpreventing an inflammation and/or for combating an inflammatory diseaseand/or an autoimmune disease. The term “inhibitor ofL-α-lysophosphatidylcholine” is intended to mean a molecule (or a set ofmolecules) which blocks the inflammatory and/or immunostimulant activityof the L-α-lysophosphatidylcholine, in particular by blocking thedifferentiation of mature dendritic cells byL-α-lysophosphatidylcholine. It is also intended to mean a module havingeffects opposite to those of L-α-lysophosphatidylcholine, by modulatingin particular the PPARδ/PPARγ ratio. By way of indication, mention maybe made of a lipid emulsion comprising triglycerides, phospholipids andglycerol, such as in particular INTRALIPID®, LIPOID E-80®, a PPARgammaagonist. PPARgamma agonists are well known to those skilled in the art,and mention may be made, in a nonlimiting manner, of molecules of thethiazolidinedione class, such as in particular ciglitazone,troglitazone, pioglitazone, rosiglitazone, etc. Such inhibitors couldthus be used in vivo, for producing a medicinal product for decreasingan inflammatory response, in particular an inflammatory response of thejoints, or especially during a transplant. According to a preferredembodiment of the invention, the inhibitor ofL-α-lysophosphatidylcholine is a lipid emulsion comprisingtriglycerides, phospholipids and glycerol. Preferably, the triglyceridesare extracted from plant oil such as in particular soybean oil. Evenmore preferably, the lipid emulsion comprises from 15 to 25% of soybeanoil, preferably 20% of soybean oil, from 0.5 to 1.5% of eggphospholipids, preferably 1.2%, and from 1.8 to 2.6% of glycerol,preferably 2.2% of glycerol. According to another preferred embodimentof the invention, the inhibitor is a PPARgamma agonist as defined above.

As inflammatory disease, mention may be in particular be made ofsarcomatosis, lupus, rheumatoid arthritis, spondylarthritis, uveitis,etc.

As autoimmune disease, mention may in particular be made of type 1diabetes, multiple sclerosis, psoriasis, contact hypersensitivities,rheumatoid arthritis, spondylarthritis, etc.

FIG. 1 shows the titration curves giving the absorbance (Abs) as afunction of the logarithm of the dilution of the serum of a batch ofmice which are given LPC mixed with hen egg lysozyme (HEL, 1 mg/ml), orHEL alone.

The following examples are given by way of explanation and are in no waylimiting in nature. They will make it possible to understand theinvention more thoroughly.

EXAMPLE 1 Differentiation of Monocytes in Culture into Dendritic Cellsin the Presence or Absence of L-α-lysophosphatidylcholine (LPC)

Isolation, placing in culture and initiation of the differentiation ofmonocytes—The monocytes are isolated from human peripheral blood bymeans of first density gradient centrifugation (620 g; 20 minutes) inFicoll-Hypaque, followed by second centrifugation (770 g; 20 minutes) ina 50% Percoll solution. The monocytes are then purified byimmunomagnetic depletion (Dynal, Oslo, Norway), using a cocktail ofanti-CD19 (hydridoma 4G7) (Becton Dickinson, Francklin Lakes, N.J.,USA), anti-CD3 (OKT3, American Type Culture Collection, Rockeville, Md.)and anti-CD56 (NKH1, Beckman Coulter, Fullerton, Calif., USA) monoclonalantibodies. The monocytes thus obtained are then purified to at least90%, as shown by the absence of CD1a markers and the presence of theCD14 marker. The differentiation of the monocytes into dendritic cellsis initiated with 40 ng/ml of recombined human GM-CSF(Granulocyte-Macrophage Colony Stimulating Factor) and 250 U/ml ofrecombined human interleukin IL-4.

The monocytes are placed in culture in RPMI 1640 medium (LifeTechnologies, Rockeville, Md., USA) enriched with 2 mM of glutamine(Life Technologies), 10 mM of Hepes (Life Technologies), 40 ng/ml ofgentamycin (Life Technologies) and 10% of lipoprotein-depleted fetalcalf serum (LPDS, Sigma, St Quentin-Fallavier, France).

It should be noted that the culture medium presented in this example isan LPDS culture medium. Comparable results can be obtained using otherculture media, such as an FCS medium, i.e. a medium containing 10% ofnon-lipoprotein-depleted fetal calf serum, or could be obtained usingany synthetic culture medium known to those skilled in the art.

Treatment of the monocytes with LPC—5 days after the beginning of thedifferentiation of the monocytes, 40 μM of LPC(L-α-lysophosphatidylcholine; Sigma, St Quentin Fallavier, France) areadded to the culture medium for 24 hours. Control cells are alsoobtained in the absence of LPC in the culture medium.

“Control” cells and “LPC” cells are then obtained.

EXAMPLE 2 Phenotype of Cells Obtained According to Example 1

The cells used in this analysis are those obtained on the 6th day ofdifferentiation according to the protocol described in example 1.

The phenotype of the “control” and “LPC” cells is analyzed by flowcytometry on a FACSCalibur (Becton Dickinson, Francklin Lakes, N.J.,USA) using FITC (fluorosceine isothiocyanate)-conjugated anti-CD14,anti-HLA-DR and anti-CD80 and PE (phycoerythrin)-conjugated anti-CD1a,anti-CD83, anti-CD86 and anti-CD40 (Beckman Coulter). According to thestate of the art, monocytes preferentially have a CD14+ CD1a− phenotype,immature dendritic cells preferentially have a CD14− CD1a+CD86−phenotype, and mature dendritic cells preferentially have a CD14− CD1aintermediate—CD86+ phenotype.

The results obtained are given in table 1.

TABLE 1 Expression of the CD83, HLA-DR and CD86 markers in the absence(control) or in the presence of LPC CD83 HLA-DR CD86 Control 5.79 78.76117.77 LPC 11.74 146.31 759.69

The “LPC” cells obtained after initiation of the differentiation of themonocytes in the presence of LPC exhibit a phenotype comparable to thatof mature dendritic cells, as demonstrated in particular by theinduction of CD86 markers and the increase in HLA-DR compared with thephenotype of the “control” cells.

EXAMPLE 3 Internalization Capacity of the Cells Obtained According toExample 1

The “control” and “LPC” cells used in this analysis are those obtainedafter 6 days of differentiation according to the protocol described inexample 1. These cells are incubated at 37° C.:

-   -   for 30 minutes with 1 mg/ml of FITC-T70-Dextran (Sigma) in order        to estimate the capacity of these cells for internalization by        endocytosis,    -   for 30 minutes with 1 mg/ml of Lucifer Yellow (ref. L0259,        Sigma, St Quentin-Fallavier, France) in order to estimate the        capacity of these cells for internalization by pinocytosis,    -   for 3 hours with carboxylate-modified yellow-green FluoSpheres        (trade name, 0.45 μm, Molecular Probes, Leiden, The Netherlands)        in order to estimate the capacity of these cells for        internalization by macropinocytosis.

The internalization is stopped on ice with a cold PBS buffer containing0.1% of BSA (Bovine Serum Albumin) and 0.05% of NaN₃. The “control” and“LPC” cells are washed 3 times at 4° C. in this same buffer and thefluorescence is quantified by FACScalibur (trade name, BectonDickinson).

As shown in table 2, the capacities for internalization by endocytosis,pinocytosis and macropinocytosis are greatly decreased by the additionof LPC to the culture medium on the 5th day of differentiation of themonocytes, compared to the control cells differentiated in the absenceof LPC.

TABLE 2 Capacity for internalization by endocytosis, pinocytosis andmacropinocytosis of the monocytes differentiated in the absence(control) or in the presence of LPC Endocytosis PinocytosisMacropinocytosis Control 100% 100% 100% LPC 53% 49% 66%

The decrease in internalization capacities is one of the characteristicsof mature dendritic cells. These results show that, in the presence ofLPC, the monocytes have a strong tendency to differentiate into maturedendritic cells.

EXAMPLE 4 Ability of the Cells Obtained According to Example 1 toStimulate T Lymphocytes

The “control” and “LPC” cells used in this analysis are those obtainedafter 6 days of differentiation according to the protocol described inexample 1.

Naïve allogenic T lymphocytes are isolated from human peripheral blood.Peripheral blood mononuclear cells are isolated by density gradientcentrifugation (600 g, 20 minutes) in the presence of Ficoll-Hypaque.After elimination of the monocytes on a Percoll gradient, the peripheralblood lymphocytes are found in the dense fraction. The T lymphocytes arepurified by immunomagnetic depletion using a cocktail of anti-CD19(antibody 4G7) (Becton Dickinson, Francklin Lakes, N.J., USA), anti-CD16(antibody 3G8), anti-CD56 (antibody NKH1), anti-glycophorin A (antibody11E4B7.6) and anti-CD14 (antibody RMPO52), monoclonal antibodies sold byBeckman Coulter.

The purified T lymphocytes are cultured in flat-bottomed 96-well cultureplates with the “control” or “LPC” cells.

2×10⁵T cells are cultured in 200 μl of culture medium according to amonocytes differentiated in the presence or absence of LPC/T cells ratio(DC/LT ratio) of 1:5, 1:10 or 1:20. After 4 days, 50 μl of the culturesupernatant are used to determine the secretion of IL-2 (interleukin 2)and of γIFN (gamma interferon) using an ELISA kit (Endogen, Woburn,Mass., USA).

As shown in table 3, the secretion of IL2 and of γIFN by the Tlymphocytes, conventionally expressed according to the dendritic cells/Tlymphocyte ratio (DC/LT ratio) is greatly stimulated by the cellsoriginating from the differentiation of monocytes in the presence of LPCadded 5 days after the initiation of monocyte differentiation (“LPC”cells), compared to the control results (“control” cells).

TABLE 3 Stimulation of the secretion of IL2 and of γIFN by Tlymphocytes, induced by the monocytes differentiated in the absence(control) or in the presence of LPC DC/LT ratio 0 0.05 0.1 0.2 ControlIL-2 0 6 ± 8 15 ± 6  35 ± 11 γIFN 0 41 ± 14 51 ± 4  119 ± 9  LPC IL-2 023 ± 1  47 ± 26 173 ± 31  γIFN 0 73 ± 47 439 ± 108 795 ± 19 

These results show an increase in the abilities of the monocytesdifferentiated in the presence of LPC to stimulate allogenic Tlymphocytes, which is a characteristic of mature dendritic cells. By wayof indication, comparable results are obtained whether the LPC isdissolved in ethanol or is in the form of a lipid emulsion.

EXAMPLE 5 Properties of the Cells Obtained According to Example 1:Comparison with Mature Dendritic Cells Obtained by Differentiation ofMonocytes in the Presence of Oxidized LDLs

The aim of this example is to demonstrate that the mechanisms of actioninvolved in the differentiation of monocytes into mature dendritic cellsin the presence of LPC could be different from those involved in thedifferentiation of monocytes into mature dendritic cells in the presenceof oxidized LDLs (Perrin-Cocon et al. The Journal of Immunology, 167:3785-3891, 2001).

In this example, the cells used are “control” and “LPC” cells asobtained in example 1, and also “LDLox” cells obtained as described inthe scientific publication (Perrin-Cocon et al. The Journal ofImmunology, 167: 3785-3891, 2001), i.e. after differentiation of themonocytes in culture in the presence of oxidized LDLs added to themedium 5 days after the initiation of differentiation.

The inventors investigated whether the inhibitors of the action ofoxidized LDLs on the differentiation of monocytes into mature dendriticcells also inhibited the action of LPC on the differentiation ofmonocytes into mature dendritic cells.

Thus, a solution of INTRALIPID® (50 μg/ml of phospholipids, FreseniusKabi, Sèvres, France), or a solution of synthetic molecules that arelipid in nature (LIPOÏD E-80®, Lipoïd Ag, Ludwigshafen, Germany, 50μg/ml of phospholipids) was added to the culture medium on the 5th dayof differentiation. The expression of CD86 by the “LPC” and “LDLox”cells in the presence of these inhibitors is analyzed according to theprotocol described in example 2, and the results are given in table 4.

TABLE 4 Inhibition (in %) of the expression of CD86 by the LCP and LDLoxcells, by Intralipid ® and lipoïd E-80 Intralipid ® Lipoïd E-80 LDLox88% ± 10 81% ± 14 LPC 86% ± 12 24% ± 19

The addition of INTRALIPID® inhibits the differentiation of monocytesinto mature dendritic cells by means of oxidized LDLs and by means ofLPC, in a comparable manner (inhibition of the order of 80%).

On the other hand, surprisingly, the addition of LIPOÏD E-80®includes an81% inhibition of the expression of CD86 when the monocytes are culturedin the presence of oxidized LDLs, although this inhibition is only 24%when the monocytes are cultured in the presence of LPC.

These results suggest that the mechanisms of action of LPC for thedifferentiation of monocytes into mature dendritic cells is differentfrom the mechanisms of action of oxidized LDLs.

EXAMPLE 6 Cellular Mechanisms Involved in the Differentiation ofMonocytes into Mature Dendritic Cells in the Presence of LPC

In order to advance further in the search for the cellular mechanisms ofthe action of LPC in the differentiation of monocytes into maturedendritic cells, the inventors investigated which receptors could beinvolved.

Initially, in order to determine whether the receptors involved in theaction of LPC in the differentiation of monocytes into mature dendriticcells belonged to the family of G protein-coupled receptors, PTX(pertussis toxin; 100 ng/ml), known to block Gi proteins, was added tothe culture medium for 3 hours. The culture medium was then changed andthe LPC was added as described in example 1.

The results given in table 5 show that preincubation of the monocyteswith PTX blocks the increase in CD86 induced by LPC, suggesting that theaction of LPC involves Gi protein-coupled receptors.

TABLE 5 Inhibition of the expression of CD86 by the “LPC” cells, withPTX Induction of CD86 LPC 100% LPC + PTX 10%

These receptors could in particular be the following receptors:

-   -   G2A receptor (G2A-R): it has recently been demonstrated that LPC        is a high-affinity ligand for the G2A protein, a G protein        coupled to a receptor expressed in lymphocytes. In addition, the        stimulation of G2A by LPC induces phosphorylation of an        extracellular kinase (ERK1/2: extracellular signal-related        kinases). This phosphorylation can, moreover, be observed when        LPC is added to the culture medium, suggesting the involvement        of the G2A receptor in the differentiation of monocytes into        mature dendritic cells by means of LPC,    -   PAF receptor (PAF-R) as described above; certain effects of LPC        could involve the PAF receptor in various types of cells,    -   GPR4 receptor: LPC is also a ligand for the GPR4 protein,        another G protein coupled to a receptor having a high affinity        for sphingosylphosphorylcholine.

Next, the inventors investigated the transcription factors involved inthe maturation process induced by LPC. In order to determine whetherPPARs are involved in the LPC-induced maturation, the ability of the twotranscription factors PPARγ and PPARδ to bind was analyzed using the gelshift technique. 3 isotypes of PPAR nuclear receptors (peroxisomeproliferator-activated receptor) exist: α, γ and δ. PPARγs are thetargets for molecules of the thiazolidinedione class, such asciglitizone, used in the treatment of type II diabetes. PPARδs have abroader expression and can be repressors of PPARγs. The monocytesundergoing differentiation were incubated for two hours with a solutionof LPC (40 μM) (“LPC” cells). “Control” cells are also obtained in theabsence of LPC in the culture medium. After having harvested the cells,the nuclear proteins were extracted with the Nuclear extract Kit(Sigma). The nuclear proteins were then brought into contact with aratioactively labeled probe containing a response element recognized byPPARs. After migration on a nondenaturing gel, a band containing PPARγand a doublet containing PPARδ were identified using antibodies, by theSupershift technique. The PPAR binding activity was determined bymeasuring the intensity of these bands. These activities are expressedas a percentage with respect to the “control” cells.

TABLE 6 Modulation of the activity of PPARγ and PPARδ by LPC PPARγ PPARδControl 100% 100% LPC 0% 263%

The treatment with LPC induces a large decrease in the binding activityof PPARγ, which can result in complete disappearance, and greatlystimulates the activity of PPARδ.

The inventors then used a PPARγ agonist, ciglitizone. A solution ofciglitizone (Sigma, 50 μM) was added to the culture medium on the fifthday of monocyte differentiation, as described in example 1, 15 minutesbefore the addition of a solution of LPC (40 μM, for 2 hours). The cellsobtained are “LPC+ciglitizone” cells. “Ciglitizone” cells were obtainedaccording to the same protocol, but in the absence of LPC, and “LPC”cells were obtained as described in example 1, in the absence ofciglitizone.

The activity of the PPARγ and PPARδ transcription factors was measuredin these cells by the gel shift technique, as described above.

TABLE 7 Ciglitizone blocks the inactivation of PPARγ and reduces theLPC-induced increase in PPARδ PPARγ PPARδ Control 100% 100% Ciglitizone237% 106% LPC + ciglitizone 115% 147%

These results indicate that ciglitizone, which activates PPARγ, greatlyinhibits the effect of LPC on PPARγ and PPARδ, and as a result blocksthe LPC-induced maturation of dendritic cells.

The functional ability of these cells (“control”, “ciglitazone” and“LPC+ciglitazone” cells) to stimulate T lymphocytes (LT) were thenanalyzed. These cells were cultured as described in example 4, in thepresence of purified allogenic T lymphocytes. The secretion of γIFN wasmeasured as in example 4.

TABLE 8 Mixed leukocyte reaction: measurement of the dendriticcell-induced secretion of γIFN by T lymphocytes DC/LT ratio 0 0.05 0.10.2 Control 0 54 ± 82 69 ± 4  364 ± 27  LPC 0 750 ± 184 899 ± 124 1326 ±248  LPC + 0 93 ± 7  169 ± 105 484 ± 229 ciglitizone

All these results emphasize an important role for PPARs in theLPC-induced maturation of dendritic cells and also the importance of thePPARγ/PPARδ ratio in the production of functionally mature dendriticcells. This ratio is modulated by LPC and ciglitizone.

EXAMPLE 7 Differentiation of Monocytes into Mature Dendritic Cells inthe Presence of Compounds Equivalent to LPC

The inventors determined whether compounds equivalent to LPC, i.e.involving the same cellular mechanisms of action via the same membraneor intracellular receptors, could also induce the differentiation ofmonocytes into mature dendritic cells.

For this, the inventors investigated whether the addition of PAF to theculture medium could increase the action of LPC on the differentiationof monocytes into mature dendritic cells.

Thus, a solution of PAF (Sigma, 5 μM) was added to the culture medium onthe 5th day of monocyte differentiation. The expression of CD86 by the“control” cells and by the “LPC” cells, obtained in the presence or inthe absence of PAF, was analyzed according to the protocol described inexample 2, and the results are given in table 9.

TABLE 9 Expression of the CD86 marker (mean fluorescence intensity) bythe “control” and “LPC” cells in the presence or in the absence of PAFCD86 Control 49.02 PAF 102.88 LPC 287.11 LPC + PAF 676.88

These results suggest that PAF alone induces a slight overexpression ofCD86, unlike LPC, which induces a 470% increase in the expression ofCD86. On the other hand, it is important to note that PAF acts insynergy with LPC, since the action of the two compounds induces a 1200%increase in the expression of CD86.

In order to reinforce the idea that the PAF receptor is involved, theinventors studied the action of an antagonist for this receptor on thedifferentiation of monocytes into mature dendritic cells by means ofLPC.

Thus, a solution of the PAF antagonist BN52021 (Biomol, PlymouthMeeting, USA, 100 μM) was added to the culture medium on the 5th day ofmonocyte differentiation. The expression of CD86 by the “control” and“LPC” cells, obtained in the presence or in the absence of BN52021, wasanalyzed according to the protocol described in example 2, and theresults are given in table 10.

TABLE 10 Expression of CD86 by the “LPC” cells in the presence of theantagonist BN52021 CD86 LPC 100% LPC + BN52021 54%

These results suggest that the action of LPC clearly involves the PAFreceptor, but also suggest that the action of LPC could involve otherreceptors.

By way of indication, the inventors also demonstrated that theantagonist BN52021, added to the culture medium on the 5th day ofdifferentiation of the monocytes into mature dendritic cells induced byoxidized LDLs, induced a 100% inhibition of the action of the oxidizedLDLs, suggesting here again that the mechanisms of action of LPC and ofoxidized LDLs are different.

EXAMPLE 8 In Vivo Stimulation of the Immune Response Against an Antigen,by LPC

The aim of this example is to show that LPC is a molecule which is anadjuvant of the immune system and which can be used in the context ofimmunization in order to increase the specific T response against anantigen.

LPC is an inflammatory product—In this example, a solution of 100 to 500nmol of LPC dissolved in 50 μl of PBS is injected into the plantar footpad of BALB/c mice (Charles River Laboratories) on day 0. The plantarfoot pads are measured using a micrometer before and after injection, upto the 10th day, and compared to the plantar foot pads of “control”mice, obtained by injection of PBS (50 μl). The intensity of theinflammation is reflected by the thickening of the foot. The maximumthickening is observed on the 1st day, 24 h after the injection.

TABLE 11 Inflammation of the plantar foot pad induced by LPC-thickeningof the foot after 24 h (in mm) Thickening of the foot (mm) PBS 0.025 LPC100 nmol 0.35 LPC 200 nmol 0.55 LPC 300 nmol 0.725 LPC 400 nmol 1.125LPC 500 nmol 1.075

These results show that LPC induces an inflammation dependent on thedose injected.

In order to show that LPC induces the maturation and therefore themigration of dendritic cells to the draining lymph nodes, in vivo, asolution of LPC (0.1 M in dibutyl phthalate) was applied to the skin ofthe ears of BALB/c mice (Charles River Laboratories), 10 minutes beforeapplication of a solution of FITC (Sigma) at 1.5% in 1:1 dibutylphthalate/acetone. This fluorescent label is taken up by the dendriticcells of the skin. After 24 h, the auricular and maxillary lymph nodesare removed and the cells are placed in suspension. The cell suspensionis enriched in dendritic cells by metrizamide (Sigma) gradientcentrifugation. The cells are labeled with an antibody (Pharmingen)which recognizes Major Histocompatibility Complex (MHC) class Imolecules and analysis by flow cytometry makes it possible to quantifythe percentage of large cells (dendritic cells) expressing the MHC classII molecules and containing FITC. These cells are presenting cells whichhave taken up the FITC in the periphery and have migrated to the lymphnodes.

TABLE 12 Stimulation of dendritic cell migration by LPC FITC⁺ dendriticcells FITC  7.1 ± 1.7% LPC + FITC 19.3 ± 3.3%

These results show that the application of LPC stimulates the migrationof dendritic cells of the skin to the draining lymph nodes.

LPC stimulates the T response specific for a co-injected solubleantigen. LPC dissolved in PBS (250 or 500 nmol in 50 μl) is mixed withhen egg lysozyme (HEL, 50 μg) and this mixture is injected into theplantar foot pad of BALB/c mice. After 7 days, the popliteal lymph nodesare removed and their cells are restimulated in triplicate in vitro, ina culture medium containing 30 μM of HEL antigen, or in the absence ofHEL. After 3 days, the T lymphocyte proliferation is measured byincorporation of triturated thymidine for 16 h.

TABLE 13 Proliferation of HEL-specific T cells (incorporation oftriturated thymidine in CPM; mean of triplicates) ProliferationImmunization in the absence Restimulation conditions of HEL with HEL (30μM) HEL 1315 ± 275  5370 ± 1983 HEL + LPC 250 nmol 1608 ± 7 10885 ± 2861HEL + LPC 500 nmol 2101 ± 87 23739 ± 4265

These results show that LPC co-injected with the antigen promotes theactivation in vivo of T lymphocytes specific for this antigen.

LPC stimulates the production of specific antibodies against aco-injected soluble antigen. LPC dissolved in PBS (350 nmol in 50 μl) ismixed with hen egg lysozyme (HEL, 1 mg/ml) and this mixture is injectedinto the plantar foot pad of BALB/c mice. The “HEL+LPC” batch of micereceives 50 μl of this mixture in the foot pads of the two hind feet.The “HEL” batch of mice receives 50 μl of HEL solution (1 mg/ml in PBS)in the two feet. After 15 days, a booster injection is givensubcutaneously on the 2 flanks. The “HEL+LPC” batch receives, on eachflank, 100 μl of a solution of LPC (5 mM) and of HEL (1 mg/ml) in PBS.The “HEL” batch receives, on each flank, 100 μl of a solution of HEL (1mg/ml) in PBS. Two weeks later, blood is taken from the mice and theIgGs specific for the HEL antigen are assayed by ELISA with respect to astandard IgG solution range. The titration curves giving the absorbance(Abs) as a function of the logarithm of the serum dilution arerepresented in FIG. 1.

These results suggest that LPC co-injected with the antigen promotesactivation of the immune system and induces the synthesis of antibodiesspecific for the antigen, whereas injection of the antigen alone doesnot induce a response. LPC induces a humoral response against theantigen, demonstrating its adjuvant capacity.

LPC can induce a CD8+ T lymphocyte response in vivo. In this example, atest for delayed contact hypersensitivity to haptens was used. In thismodel, BALB/c mice are sensitized by application of a hapten, DNFB(1-fluoro-2,4-dinitrobenzene, 0.5% solution in 1:1 olive oil/acetone),to the back. Five days later, a non-irritant dose of DNFB (0.2% solutionin 1:1 olive oil/acetone) is applied to the left ear, whereas the rightear receives the solvent. In this elicitation phase, CD8⁺ T lymphocytesspecific for the haptenized proteins are recruited and infiltrate theear, secreting γIFN and producing an edema.

A “control” batch of mice was sensitized with DNFB alone and anotherbatch (“LPC” mice) received 500 nmol of LPC by application to the skinof the back of 20 μl of an ethanolic solution of LPC, 10 minutes beforethe application of DNFB. Five days later, the two batches were treatedin the same way for the elicitation phase and the thickening of the earswas measured using a micrometer 48 h after this application.

TABLE 14 Measurement of the ear edema Thickening of the Thickening ofthe right ear (μm) left ear (μm) Control 0 ± 4  67 ± 32 LPC 0 ± 3 149 ±31

These results show that the application of LPC 10 minutes before thehapten during the sensitization phase increases the edema induced duringthe 2nd application of the hapten (elicitation). This means that LPCstimulates the response relayed by CD8+ T lymphocytes.

EXAMPLE 9 Action of a Lipid Emulsion Such as INTRALIPID® on theDifferentiation of Monocytes into Mature Dendritic Cells

Since it was shown in example 5 that a lipid emulsion such asINTRALIPID® blocks the LPC-induced production of mature dendritic cells,the inventors also investigated whether this blocking came from anindirect action of this lipid emulsion via inhibition of the action ofLPC and/or from a direct action of this lipid emulsion.

INTRALIPID® regulates PPARs in vitro. Initially, a solution ofINTRALIPID® (50 μg/ml of phospholipids) was added to the culture mediumon the 5th day of monocyte differentiation, according to a protocolcomparable to that described in example 6. The cells were harvestedafter 1 h, 2 h or 8 h of incubation with INTRALIPID®, the nuclearproteins were extracted and the activity of PPARγ and PPARδ wasdetermined as described in example 6.

TABLE 15 Modulation of the activity of PPARγ and PPARδ of monocytesundergoing differentiation, by Intralipid ® Incubation time withIntralipid (h) PPARγ PPARδ 0 100% 100% 1 130% 94% 2 160% 94% 8 170% 77%

These results show that INTRALIPID® alone activates PPARγ and inhibitsthe activity of PPARδ. This action of INTRALIPID® is opposite to that ofLPC.

Secondly, a solution of INTRALIPID® (50 μg/ml of phospholipids) wasadded to the culture medium on the 5th day of monocyte differentiation,15 minutes before the addition of a solution of LPC (40 μM)(“LPC+Intralipid” cells). “Intralipid” cells were also obtained in theabsence of LPC. “LPC” cells were obtained in the absence of INTRALIPID®.The cells were harvested after 2 h of incubation, the nuclear proteinswere extracted and the activity of PPARγ and PPARδ was determined asdescribed in example 6.

TABLE 16 Intralipid ® blocks the LPC-induced generation of maturedendritic cells by modulating the activity of PPARγ and PPARδ PPARγPPARδ Control 100% 100% Intralipid 156% 99% LPC 1% 160% LPC + Intralipid40% 80%

INTRALIPID® also blocks the action of LPC by modulating the activity ofPPARγ and PPARδ.

INTRALIPID® blocks the inflammation and the immune response induced byLPC, in vivo. In this example, 3 batches of BALB/c mice are immunizedagainst the hen egg lysozyme (HEL) antigen by injection of 50 μl ofsolution into the plantar foot pad. The “HEL” control batch receives 50μg of HEL, the “HEL+Intralipid” batch receives a mixture of INTRALIPID®(45 μl) and of 50 μg of HEL (5 μl of a solution at 10 mg/ml). The“HEL+LPC” batch receives 50 μg of HEL with 350 nmol of LPC dissolved inPBS. The “HEL+LPC+Intralipid” batch receives 50 μg of HEL mixed with 350nmol of LPC dissolved in 45 μl of INTRALIPID®. After 24 hours, thethickness of the foot pads is measured using a micrometer, and comparedwith the measurement taken before injection. The difference in thicknessis proportional to the intensity of the inflammation.

TABLE 17 Intralipid reduces the inflammation induced in vivo by LPCAverage thickness of the foot at 24 h (mm) HEL + Intralipid 0.19 ± 0.04HEL + LPC 1.07 ± 0.19 HEL + LPC + Intralipid 0.45 ± 0.04

These results show that injecting INTRALIPID® at the same time as LPCreduces the LPC-induced inflammation of the plantar foot pad by 70%.

Seven days after the injection, the popliteal lymph nodes are removedand their cells are restimulated in vitro in triplicate, in a culturemedium containing 10 μM of HEL or in the absence of HEL. After 3 days,the proliferation of HEL-specific T lymphocytes is measured byincorporation of triturated thymidine for 16 h.

TABLE 18 Proliferation of HEL-specific T cells (mean of the mice)Proliferation Restimulation Immunization in the absence with HELconditions of HEL (cpm) (10 μM) (cpm) HEL  370 ± 212  738 ± 408 HEL +LPC 2568 ± 1570 11340 ± 4426 HEL + LPC + Intralipid 1124 ± 541  3959 ±2103

These results show that INTRALIPID® injected at the same time as LPCblocks the LPC-induced stimulation of the T response.

All these results show that INTRALIPID® blocks the LPC-inducedgeneration of mature dendritic cells by means of an indirect action ofINTRALIPID® via inhibition of the action of LPC, but also by means of adirect action of INTRALIPID® on PPARs, antagonist for LPC. These resultsshow that INTRALIPID® alone has an anti-inflammatory role.

All these results show the importance of the PPARγ/PPARδ ratio in thegeneration of functionally mature dendritic cells. This ratio ismodulated by LPC, but also, inversely, by a lipid emulsion such asINTRALIPID® or ciglitizone.

1. A method for the differentiation in vitro of monocytes into maturedendritic cells, according to which: (a) monocytes are provided in asuitable culture medium, (b) the differentiation of the monocytes intodendritic cells is induced in the presence of GM-CSF and IL-4; (c) amolecule consisting of L-α-lysophosphatidylcholine (LPC) is added tosaid medium; and (d) mature dendritic cells are obtained.
 2. The methodas claimed in claim 1, according to which, in step (c), theL-α-lysophosphatidylcholine is added to said medium at a finalconcentration in the medium of between 10 and 80 μM.
 3. The method asclaimed in claim 1, according to which, in step (c), theL-α-lysophosphatidylcholine is added to said medium between the 3rd and6th day of monocyte differentiation.
 4. The method as claimed in claim1, wherein, in step (c), at least one biological agent is also added tothe culture medium, wherein the biological agent is a bacterial, viral,yeast, parasite or fungal antigen, an autologous and/or heterologoustumor cell lystate antigen, a tumor antigen, a nucleic acid whichencodes a bacterial, viral, yeast, parasite or fungal antigen, ornucleic acid which encodes a tumor antigen.
 5. The method as claimed inclaim 1, wherein, in step (c), a platelet activating factor (PAF) isalso added to the culture medium.